OBJETIVOS DEL ÁREA DE IMPORTACIONES DE IDEAL ALAMBREC S.A Y

Maximum Likelihood

Maximum likelihood analysis yielded a tree (Figure 2.2) which shows each o f the four gibbon subgenera Nomascus, Symphalangus, Bunopithecus and Hylobates as monophyletic. This is in agreement with previous studies based on molecular,

morphological and behavioural data (e.g. Chivers, 1977; H aim off et al., 1982; Cronin et a l, 1984; Marshall and Sugardjito, 1986; Geissmann, 1993, 1995; Garza and Woodruff,

1992; Hayashi et al., 1995; Hall, et a l, 1996, 1998). The tree shows the following relationships: the subgenera Symphalangus and Bunopithecus are

successively more closely related to subgenus Hylobates. Interrelationships among taxa in the subgenus Nomascus could not be resolved using this method. Hence, the tree shows a polytomy between concolor, gabriellae and leucogenys. W ithin subgenus

Hylobates, pileatus and klossii are sister taxa, and agilis is sister taxon to {lar +

human concolor {Nomascus) gabriellae {Nomascus) leucogenys {Nomascus) syndactylus {Symphalangus) hoolock {Bunopithecus) moloch {Hylobates) agilis {Hylobates) lar {Hylobates) muelleri {Hylobates) klossii {Hylobates) pileatus {Hylobates)

Maximum likelihood assuming a molecular clock

The maximum likelihood tree constructed under the assumption o f a molecular clock (Figure 2.3) is similar to the tree constructed using maximum likelihood analysis. The tree shows that the subgenera Nomascus, Symphalangus and Bunopithecus are successively more closely related to subgenus Hylobates. Relationships within

subgenera are different in the two analyses. The tree depicted in Figure 2.3, assuming a molecular clock, shows that within the subgenus Nomascus, leucogenys is sister taxon

to {concolor + gabriellae). Within subgenus Hylobates, moloch and agilis are

successively more closely related to a clade comprising {lar + muelleri) and {klossii +

pileatus).

Under the assumption o f a molecular clock it is possible to give estimates o f the dates o f divergence at different nodes on the tree (Figure 2.3, Table 2.4). These

estimates were calibrated using the great ape-gibbon split. Dates for this divergence, however, are controversial, ranging from 12 Ma (million years ago) to 36 Ma, based on a variety o f data. Since fossil hylobatids are scarce (Tyler, 1993) evidence for their divergence from the other apes has mainly originated from the field o f molecular biology. Combined biochemical results based on analysis o f blood groups and histocompatibility antigens, chromosome banding patterns, protein structure and antigenicity, amino acid sequences o f proteins, and DNA endonuclease restriction mapping, sequencing and reassociation kinetics, indicate a great ape-gibbon split at no more that 15 Ma (see Tyler, 1993 and references therein). However, more recent molecular analyses based on the complete mitochondrial genome have pushed back the great ape-gibbon split to 36 Ma (Amason et a l, 1996). In light o f this controversy the two alternative hypotheses for the date of this split (36 Ma and 15 Ma) are employed here to calibrate possible dates o f divergence within the genus Hylobates (Table 2.4).

If the great ape-gibbon split is more ancient than previously believed this would suggest that gibbon radiation began over 20 Ma (node A, Figure 2.3). According to cytochrome b gene, this radiation involved the divergence o f taxa in the subgenus

Nomascus, from the rest o f the gibbons. A fairly prolific radiation followed between

about 20 and 10 Ma, involving syndactylus, hoolock and gibbons in the subgenus

Hylobates. Taxa in the subgenus Hylobates radiated between about 11 and 7 Ma.

According to this reconstruction, taxa in the subgenus Nomascus are a relatively more recent radiation, diverging about 4 to 1 Ma (nodes I and J, Figure 2.3).

Using the more recent date for the divergence o f great apes and gibbons, based on combined evidence, it appears that the gibbon radiation dates to approximately 10 Ma (node A, Figure 2.3). According to this reconstruction, the clade comprising

syndactylus, hoolock, and taxa in the subgenus Hylobates radiated between about 8 and

3 Ma. These estimates also indicate that taxa in the subgenus Nomascus represent a recent radiation between about 1.7 and 0.3 Ma (nodes 1 and J, Figure 2.3).These results are in agreement with previous findings based on molecular data which indicate that there was a rapid radiation o f hylobatids between approximately 1 0 - 6 Ma (Zehr et al.,

1996; Porter et al., 1997). Zehr et al. (1996) analysed cytochrome oxidase subunit 11 sequence data for various species of gibbon and estimated that there was a rapid radiation o f gibbons 6-8 Ma. Porter et al. (1997) analysed sequences o f the e-globin locus from a variety o f primate taxa including two species o f gibbon and estimated that the gibbon radiation dates to approximately 9.9 Ma. Thus, the estimated divergence dates presented in this study, based on cytochrome b gene, are compatible with previous estimates.

human — leucogenys {Nomascus) ’“‘concolor {Nomascus) — gabriellae {Nomascus) syndactylus {Symphalangus) hoolock {Bunopithecus) moloch {Hylobates) agilis {Hylobates) lar {Hylobates) muelleri {Hylobates) klossii {Hylobates) pileatus {Hylobates)

Fig u re 2.3 Maximum likelihood tree constructed under the assumption o f a molecular clock, based on cytochrome b gene sequence data. (Subgenera are indicated in

T able 2.4 Estimates o f divergence dates for hylobatids based on maximum likelihood analysis o f cytochrome b gene, assuming a molecular clock. Calibrated using two estimates o f the great ape-gibbon split

Node * Estim ates of divergence dates based on

36 M a great ape-gibbon split (A rnason et at., 1996)

M a

E stim ates of divergence dates based on

15 M a g reat ape-gibbon split (Tyler, 1993) M a A 24.7 10.5 B 20.3 8.6 C 18.2 7.7 D 11.9 5.1 E 11.4 4.8 F 10.9 4.6 G 8.8 3.7 H 7.2 3.1 I 4.2 1.8 J 0.8 0.3

* Nodes are depicted in Figure 2.3, maximum likelihood tree constructed under the assumption o f a molecular clock using cytochrome b sequence data. Representations are as follows; A = split between the clade comprising taxa in the subgenus Nomascus and the clade comprising the rest o f the hylobatids (subgenera Symphalangus, Bunopithecus,

and Hylobates), B = split between syndactylus and hoolock, C = split between hoolock

and clade comprising taxa in the subgenus Hylobates, D = split between moloch and clade comprising agilis, {lar + muelleri), and (klossii + pileatus), E = split between

agilis and clade comprising (lar + muelleri) and (klossii + pileatus), F = split between

(lar + muelleri) and (klossii + pileatus), G = split between klossii and pileatus, H = split

between lar and muelleri, I = split between leucogenys and (concolor + gabriellae), J = split between concolor and gabriellae.

Parsimony analysis

Parsimony analysis o f the cytochrome b dataset produced nine equally

parsimonious trees. Figure 2.4 shows a strict consensus tree derived from the parsimony analysis. The topology o f this tree is almost identical to the maximum likelihood

analysis and maximum likelihood assuming a molecular clock. Again all o f the subgenera are monophyletic and the tree shows the following relationships: the subgenera Nomascus, Symphalangus and Bunopithecus are successively more closely related to taxa in the subgenus Hylobates. The strict consensus tree can provide no resolution to relationships among taxa in the subgenus Nomascus. Within the subgenus

Hylobates, moloch forms a separate clade to a clade comprising agilis, {lar + muelleri)

human concolor {Nomascus) gabriellae {Nomascus) leucogenys {Nomascus) syndactylus {Symphalangus) hoolock {Bunopithecus) moloch {Hylobates) agilis {Hylobates) lar {Hylobates) muelleri {Hylobates) klossii {Hylobates) pileatus {Hylobates)

Bootstrap analysis

The bootstrap 50% majority rule consensus tree (Figure 2.5) has a similar topology to the parsimony and likelihood trees, showing the subgenera Nomascus,

Symphalangus and Bunopithecus are successively more closely related to subgenus

Hylobates. The tree shows that taxa in the subgenus Hylobates form a polytomy,

including {lar + muelleri) as sister taxa. Bootstrap support for this clade is high, 97%, with 78% support for the {lar + muelleri) group. Taxa in the subgenus Nomascus also form a polytomy, with 100% bootstrap support. Support for the clades including

67% 50% 97% 100% 78% human concolor {Nomascus) gabriellae {Nomascus) leucogenys {Nomascus) syndactylus {Symphalangus) hoolock {Bunopithecus) - moloch {Hylobates) agilis {Hylobates) lar {Hylobates) muelleri {Hylobates) klossii {Hylobates) pileatus {Hylobates)

Control Region M aximum Likelihood

Maximum likelihood analyses using the two control region datasets resulted in identical trees (Figure 2.6). The analysis yielded some unexpected results which contradict other findings (e.g. Chivers, 1977; H aim off et a l, 1982; Cronin et al., 1984; M arshall and Sugardjito, 1986; Geissmann, 1993, 1995; Garza and Woodruff, 1992; Hayashi et al., 1995; Hall, et al., 1996, 1998). The trees indicate that moloch, agilis and

pileatus form a separate clade, not only from the rest o f subgenus Hylobates, but from

all o f the other gibbons. Possible explanations for this anomaly are discussed in section 2.5. Aside from this anomaly, the clade comprising the rest o f the four subgenera indicates that lar, muelleri, hoolock, and syndactylus are successively more closely related to a clade comprising {concolor + concolor siki). Interrelationships between lar,

muelleri and the other gibbons are not resolved. Hence, a polytomy between lar and

muelleri is depicted in Figure 2.6.

Maximum likelihood (ML) analyses using control region data contradict

findings o f the ML using cytochrome b gene data (compare Figure 2.6 with Figure 2.2). ML analysis o f the control region datasets implies that the subgenus Hylobates is not monophyletic. This is on contrast to the ML analysis o f cytochrome b gene data, plus many other published studies, which suggest that subgenus Hylobates is monophyletic. (e.g. Chivers, 1977; Haimoff et a l, 1982; Cronin et a l, 1984; Marshall and Sugardjito, 1986; Geissmann, 1993, 1995; Garza and Woodruff, 1992; Hayashi et al., 1995; Hall, et a l, 1996, 1998).

Aside from this anomaly, relationships among the four gibbon subgenera also differ between the ML analysis o f cytochrome b gene and control region data. Analyses based on cytochrome b gene sequences result in a tree indicating that Nomascus,

Symphalangus, Bunopithecus are successively more closely related to taxa in the subgenus Hylobates. The analysis based on control region sequences (aside from the

moloch {pileatus + agilis) anomaly) suggests Hylobates, Bunopithecus, and

Symphalangus are successively more closely related to Nomascus. In other words the

sequence o f evolution in the tree constructed from control region data is completely the reverse o f the sequence observed in the cytochrome b dataset.

human muelleri {Hylobates) lar {Hylobates) hoolock {Bunopithecus) hoolock* {Bunopithecus) syndactylus {Symphalangus)

concolor siki {Nomascus)

concolor siki* {Nomascus)

concolor {Nomascus)

concolor* {Nomascus)

moloch {Hylobates)

pileatus {Hylobates)

agilis {Hylobates)

F ig u re 2.6 M axim um likelihood tree based on control region sequence data. The same topology was obtained for each o f the control region alignm ents, C R l and CR2. (Subgenera are indicated in brackets). * indicates a duplicate sample.

Maximum likelihood assuming a molecular clock

Maximum likelihood analyses assuming a molecular clock using the two control region datasets, resulted in two trees with different topologies (Figures 2.7 and 2.8). Both trees show moloch and {pileatus + agilis) as a separate clade to all the other gibbons. Interrelationships between the other hylobatid taxa, however, differ between the two trees. Figure 2.7 (based on the control region 1 dataset) shows a tree in which

hoolock and {muelleri + lar) form a separate clade to syndactylus and concolor. In

Figure 2.8 (based on the control region 2 dataset) concolor and concolor siki form sister taxa, and form a separate clade to {syndactylus + hoolock) and {muelleri + lar).

Divergence times were calculated using an early (36 Ma) and more recent (15 Ma) calibration date o f the great ape-gibbon split, as before. In each case the results were similar for both trees. Assuming an early great ape-gibbon split o f 36 Ma, divergence time was estimated to be between 20 and 22 Ma for the clade excluding

moloch, pileatus and agilis (node A, Figure 2.8). Divergence time, assuming a great

ape-gibbon split o f 15 Ma, was estimated to be between 8 and 9 Ma for the clade excluding moloch, pileatus and agilis (node A, Figure 2.8). These estimates should, however, be treated with great caution since a significant departure from a molecular clock has been shown for the control region in this study, based on the significant differences in the sequences o f certain closely related species o f gibbon in the subgenus

Hylobates (compared to previous molecular estimates, and the cytochrome b gene data

human hoolock {Bunopithecus) hoolock'^ {Bunopithecus) muelleri {Hylobates) lar {Hylobates) syndactylus {Symphalangus)

concolor siki {Nomascus)

concolor siki* {Nomascus)

concolor {Nomascus)

concolor* {Nomascus)

moloch {Hylobates)

pileatus {Hylobates)

agilis {Hylobates)

F ig u re 2.7 M axim um likelihood tree constructed under the assum ption o f a m olecular clock, based on the control region 1 dataset. Branch lengths are not shown. (Subgenera are indicated in brackets). * indicates a duplicate sample.

human

— concolor siki {Nomascus)

concolor siki*' {Nomascus)

— concolor {Nomascus) concolor* {Nomascus) syndactylus {Symphalangus) — hoolock {Bunopithecus) hoolock* {Bunopithecus) muelleri {Hylobates) lar {Hylobates) moloch {Hylobates) — pileatus {Hylobates) — agilis {Hylobates)

Parsimony analysis

Parsimony analyses o f the two control region datasets resulted in only one most parsimonious tree per analysis. The two trees have slightly different topologies (Figures 2.9, 2.10). Both trees agree with maximum likelihood analysis and maximum likelihood analysis assuming a molecular clock, as regards the anomalous grouping o f moloch,

agilis and pileatus. The trees differ in their grouping o f hoolock. In the tree based on the

control region 1 dataset, hoolock forms a sister group with {lar + muelleri), and together they form a separate clade to syndactylus as sister taxa to {concolor + concolor siki).

The tree based on the control region 2 dataset shows {lar + muelleri), hoolock and

syndactylus are successively more closely related to a clade comprising {concolor and

concolor siki).

The tree constructed using the control region 1 (C R l) dataset differs slightly from the tree constructed using the same dataset in the maximum likelihood analysis (compare Figure 2.9 with Figure 2.6). The parsimony analysis o f C Rl produced a tree in which {lar + muelleri) form sister taxa to hoolock, and together these form a separate clade from syndactylus and {concolor + concolor siki). The tree constructed from maximum likelihood analysis using C Rl shows hoolock forms a separate clade to {lar +

muelleri), and that hoolock is intermediate in position between {lar + muelleri) and

syndactylus.

Maximum likelihood analysis assuming a molecular clock based on the CRl dataset produced a tree which is similar to the parsimony analysis using the same dataset (compare Figure 2.9 with Figure 2.7)

The tree depicted in Figure 2.10 (based on the control region (CR) 2 dataset) has the same topology as the tree constructed using the same data in a maximum likelihood

analysis (compare with Figure 2.6). The trees show that, in the clade excluding moloch,

pileatus and agilis, lar and muelleri form sister taxa, and that {lar + muelleri), hoolock

and syndactylus are successively more closely related to a clade comprising {concolor +

concolor siki).

The tree depicted in Figure 2.10 is different, however, from the tree constructed using the same dataset in a maximum likelihood analysis assuming a molecular clock (compare with Figure 2.8). In the parsimony tree (in the clade excluding moloch,

pileatus and agilis) {lar + muelleri) form a separate clade to a clade comprising

syndactylus as sister taxon to {concolor + concolor siki) and forming a sister group to

hoolock. In the maximum likelihood tree based on the assumption o f a molecular clock

(in the clade excluding moloch, pileatus and agilis), {concolor + concolor siki) form a separate clade to a clade comprising syndactylus as sister taxon to hoolock, and forming a sister group to {lar + muelleri).

human muelleri {Hylobates) lar {Hylobates) hoolock {Bunopithecus) hoolock* {Bunopithecus) syndactylus {Symphalangus)

concolor siki {Nomascus)

concolor siki* {Nomascus)

concolor {Nomascus)

concolor* {Nomascus)

moloch {Hylobates)

pileatus {Hylobates)

agilis {Hylobates)

F ig u re 2.9 M axim um parsim ony tree based on the control region 1 dataset. (Subgenera are indicated in brackets).* indicates a duplicate sample.

human muelleri {Hylobates) lar {Hylobates) hoolock {Bunopithecus) hoolock'^ {Bunopithecus) syndactylus {Symphalangus)

concolor siki {Nomascus)

concolor siki* {Nomascus)

concolor {Nomascus)

concolor* {Nomascus)

moloch {Hylobates)

pileatus {Hylobates)

B ootstrap analysis

Bootstrap analysis o f CRl produced a tree which has the same topology as the tree constructed from parsimony analysis o f C R l (Figure 2.11). Bootstrap support for clades was typically high. There is 100% bootstrap support for the anomalous clade comprising moloch and {agilis and pileatus), and 99% support for the larger clade comprising the rest o f the gibbons. There is 100% support for the following pairs o f duplicate samples: {hoolock-^ hoolock*), {concolor + concolor*) and {concolor siki +

concolor siki*). There is also 100% support for {concolor + concolor siki) as sister taxa.

There is high support, 99%, for the {lar + muelleri) sister group. Finally, there are low bootstrap values for the clade grouping {lar + muelleri) and hoolock (38%), and for the clade grouping syndactylus and {concolor + concolor siki) (44%).

Bootstrap analysis o f CR2 produced similarly high bootstrap values for the same clades as C R l. The bootstrap 50% majority rule consensus tree (Figure 2.12) has the same topology as the parsimony tree constructed using CR2. Again there is 100% bootstrap support for the clade which separates moloch and {agilis + pileatus) from the rest o f the gibbons. The tree shows there is 97% support for the clade separating the other gibbons. As with the C Rl analysis, there is 100% support for the clades comprising duplicate samples, and for the clade which groups {concolor + concolor

siki) as sister taxa. There is 98% support for the grouping o f {lar + muelleri) as sister

taxa. Again there is low bootstrap support for the clades which depict relationships between hoolock, syndactylus, {lar + muelleri) and {concolor + concolor siki); 52% for the clade that separates hoolock from syndactylus, and {concolor + concolor siki), and 60% for the clade that separates syndactylus from {concolor + concolor siki).

These bootstrap values indicate that for the control region there is very high support for the anomalous separation o f moloch, agilis and pileatus from the rest o f the

gibbons. In addition, there is high support for all o f the duplicate samples indicating that systematic sequencing error was not a problem. Furthermore, consistently high

bootstrap support for the grouping o f {concolor + concolor siki) and {lar + muelleri)

indicates these taxa are closely related, and provides evidence to suggest that sequencing errors were minimal. The low bootstrap values for clades comprising

hoolock, syndactylus, {lar + muelleri) and {concolor + concolor siki) indicate that these

Discussion

Maximum likelihood analysis, maximum likelihood analysis assuming a molecular clock, parsimony and bootstrap analyses o f cytochrome b sequence data agree on the general relationship among hylobatid subgenera. Trees produced from all these types o f phylogenetic analysis indicate that Nomascus, Symphalangus, and

Bunopithecus are successively more closely related to Hylobates, and that the subgenera

are monophyletic. Interrelationships among taxa within subgenera differed between trees. Maximum likelihood analysis, parsimony and bootstrap analysis could not resolve relationships among taxa in the subgenus Nomascus. All the analyses based on

cytochrome b sequence data indicate that lar and muelleri form sister taxa, and this is supported by a high bootstrap value. Maximum likelihood and parsimony analyses also indicate a sister taxon relationship between klossii and pileatus. The relationships o f these two sister groups, (Jar + muelleri) and (klossii + pileatus), to agilis and moloch

varied between the four analyses, although most trees indicate that moloch forms a separate clade to the other taxa in subgenus Hylobates.

Re-analysis o f 11 published cytochrome b gene sequences is able to resolve relationships among the four gibbon subgenera. This is contrary to Hall et al. (1996,

1998) who concluded that they could not resolve relationships among gibbon subgenera

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